Antibody and T cell receptor production relies on a gene assembly process called V(D)J recombination, which occurs at six specific locations in the genome. Children born with a defect at any step of this gene rearrangement process have severe combined immune deficiency (SCID). In previous cycles of this grant, we have determined that hairpin opening is due to Artemis:DNA-PKcs;determined that phosphorylation is the basis for Artemis activation by DNA-PKcs;developed a widely-used human V(D)J recombination assays system;identified the first human mutations in the RAG1 and RAG2 genes, which accounts for 15% of human SCID;identified coding end effects on the efficiency and diversity of coding joint formation;determined the basis for the bias toward deletion rather than inversion in D to J joining;identified the ligase complex (XRCC4:DNA ligase IV) for the final step of V(D)J recombination using both biochemistry and genetics;created a human cell line knockout of this ligase;provided the first genetic evidence for pol X polymerases in rejoining;and developed an in vitro reconstitution of NHEJ that includes TdT, pol mu and pol lambda. Here we propose a series of studies to extend this mechanistic understanding of V(D)J recombination and determine how it contributes to human SCID. Aim 1A is focused on determining the contribution of the hairpin formation to the human immune repertoire. Aims 1B-E focus on the transition from the RAG complex to the NHEJ phase of V(D)J recombination. Aim 2 is directed at determining if RAG-generated DMA ends can activate the Artemis:DNA-PKcs complex and the phosphorylation of sites on Artemis. Aim 3 is focused on the physical and functional interaction between the Ku:DNA complex and polymerases mu and lambda. Aim 4 is directed at unifying the hairpin formation and hairpin opening steps with an NHEJ rejoining system to yield an in vitro reconstitution of V(D)J recombination using fully purified proteins. Aim 5 is focused on understanding the interplay between Artemis and DNA-PKcs using transgenic and knock-in mice. These studies will help us understand the role of DNA-PKcs in Artemis activation. Aim 6 is focused on understanding the biochemical basis for the Artemis mutations in human SCID patients. In the long-term, all of these studies will help establish optimal therapy for many human SCID patients and to understand the human immune repertoire.